Directed cell migration or chemotaxis in response to chemokine gradient is involved in development, immune function, angiogenesis as well as pathological processes associated with inflammation and cancer cell metastasis. However, little is known about molecular signaling mechanisms that cause a stationary cell to become motile. Ultimately, these signals must target the actin-myosin cytoskeleton of the cell to induce an asymmetrical polarized morphology with a leading pseudopodium and a trailing tail or rear. Pseudopodia growth is associated with actin polymerization, membrane ruffling, and formation of new focal adhesions and occurs independent of cell body translocation. On the other hand, establishment of the rear component of a polarized cell occurs after pseudopodium formation and is characterized by loss of focal adhesions and strong actin-myosin contraction. Although it is clear that these events are important for cell movement, mechanisms of gradient sensing and the temporal and spatial organization of signals that control front-rear polarity are poorly understood. This is because it is technically difficult to separate the front and back of polarized cells for biochemical analysis. We recently published a method for purification of the leading pseudopodium and rear compartments of cells polarized towards a chemoattractant gradient. In this report, we demonstrate that it is possible to specifically isolate pseudopodia undergoing membrane extension or retraction. Biochemical comparison of actively extending and retracting pseudopodia revealed striking differences in the localization and activity of several cytoskeletal-associated signaling molecules including the Rho family of GTPase, which control this process. Our findings indicate that cell polarity requires redistribution and local activation of cytoskeletal-associated signals to different poles of the migrating cell. Our preliminary work indicates that ERK and RheA differentially control pseudopodia growth and retraction in response to a chemoattractant I gradient, respectively. Using this novel biochemical fractionation method and time-lapse imaging of pseudopodia dynamics, studies outlined in this proposal will examine how G protein coupled receptors initiate the spatiotemporal organization and regulation of ERK and RheA as well as their downstream effectors during Imorphological polarization. Findings from these studies are physiologically important to understanding at the Imolecular level-how cells sense their extracellular environment and migrate dung immune responses, wound healing and cancer.